Mammalian cells need to maintain proper oxygen homeostasis in order to execute their aerobic metabolism and energy generation. Since the discovery of the hypoxia-inducible factor (HIF)-1, signaling mechanisms underlying oxygen-sensing by HIF transcription factors have been extensively studied in biological contexts (Wang et al., Redox Rep. 1996 April; 2(2):89-96). HIFs, composed of oxygen-labile α and constitutively expressed β subunits, drive the transcription of numerous genes involved in diverse cellular processes including erythropoiesis, angiogenesis, energy metabolism, ischemia, and inflammation (Semenza et al., J. Biol. Chem. 1994 Sep. 23; 269(38):237357-63 and Eltzschig et al., Nat. Rev. Drug Discov. 2014 November; 13(11):852-69). HIF is present in cells almost exclusively in two forms: HIF-1 and HIF-2. They are heterodimeric transcription factors consisting of a constitutively produced highly abundant HIF-β subunit and either a HIF-1α or HIF-2α partner, in the case of HIF-1 and HIF-2, respectively, sharing 48% sequence homology (Rabinowitz M H, J. Med. Chem. 2013 Dec. 12; 56(23):9369-4025). HIF-1 is frequently associated with metabolic and vascular responses to hypoxia, whereas HIF-2 is associated with vascular systems but also somewhat more with erythropoiesis (Ratcliffe P J, J. Clin. Invest. 2007 April; 117(4):862-5).
The mechanism by which oxygen controls HIF-1α has been revealed by the identification of HIF prolyl hydroxylases (PHDs) (Bruick and McKnight, Science 2001 Nov. 9; 294(5545):1337-40). Under normoxia, PHD hydroxylates proline residues in the oxygen dependent degradation (ODD) domain of HIF-1α, thereby allowing binding to von Hippel Lindau protein (pVHL)-elonginB-elonginC, leading to active ubiquitination and degradation with a half-life of approximately 5 min. On the other hand, the oxygen deprivation under hypoxia impairs hydroxylation of HIF-1α, by PHDs, resulting in reduced HIF-1α, turnover and subsequent induction of target gene transcription (Rabinowitz M H, J. Med. Chem. 2013 Dec. 12; 56(23):9369-4025). PHDs belong to the family of the dioxygenase enzymes that require oxygen, iron, and 2-oxyglutarate (2-OG) for their catalytic activity. Their low affinity to oxygen, which is about 2 to 10 times higher than physiological oxygen concentrations, enables the enzymes to act as oxygen sensors. Three PHD isoforms (PHD1, PHD2, and PHD3) have been identified, and their substrates are known to be quite diverse and isoform-specific (Rabinowitz M H, J. Med. Chem. 2013 Dec. 12; 56(23):9369-4025 and; Eltzschig et al., Nat. Rev. Drug Discov. 2014 November; 13(11):852-69).
PHD2 is considered critical in regulating the HIF pathway. Specifically, enhanced angiogenesis, and increased levels of vascular endothelial growth factor (VEGF)-A and erythropoietin (EPO) were observed in conditional knockout of PHD2 (Takeda et al., Circulation 2007 Aug. 14; 116(7):774-81). Such observations, along with the previous report that HIF enhanced EPO release and concomitantly increased erythropoiesis, imply that activation of HIF by modulating PHDs could be beneficial for patients with anemia and ischemia-related diseases. Accordingly, pharmacological approaches to activate the HIF pathway by inhibiting PHD activity have been pursued to treat systemic and local diseases the treatment of which can benefit from HIF activation (Rabinowitz M H, J. Med. Chem. 2013 Dec. 12; 56(23):9369-4025 and; Eltzschig et al., Nat. Rev. Drug Discov. 2014 November; 13(11):852-69).
Hypoxia and inflammation are intimately linked on many levels and have functional roles in many human diseases. Indeed, a wide range of clinical conditions is characterized by hypoxia- or ischemia-driven inflammation or by inflammation-associated hypoxia. Accumulating evidence shows that inflammatory lesions are characterized by the occurrence of tissue hypoxia that is probably a result of increased metabolism and diminished oxygen supply. For example, this is the case during the intestinal inflammation observed in patients suffering from inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease (Karhausen et al., J. Clin Invest. 2004 October; 114(8):1098-106). Such inflammatory hypoxia is caused by dramatic shifts in metabolic supply and demand ratios. Emerging evidence indicates that concomitant inhibition of PHDs and subsequent stabilization of HIFs during tissue hypoxia could function as an endogenous adaptive response to counterbalance hypoxia-driven inflammation and to restore normal cellular functions (Eltzschig et al., Nat. Rev. Drug Discov. 2014 November; 13(11):852-69).
Neuronal damage secondary to brain injuries such as cerebral hypoxia and neurodegenerative process, is a complex process that involves inflammatory changes. The activation of a common mechanism related to survival or cell death, mediated by the stabilization and trans-activation of HIF-1α, has been observed in these conditions. PHDs are the gatekeepers for the oxygen-dependent degradation of HIF-1α and also function as integrated sensors of cellular metabolism (Aragonés et al., Cell Metab. 2009 Jan. 7; 9(1):11-22). The phenomenon that hypoxic preconditioning (HP) protects against subsequent severer anoxia was discovered approximately two decades ago. Subsequently, the effects of HP have been studied intensively in vitro and in vivo models of acute hypoxia. Although the exact mechanisms are not completely disclosed, the underlying molecular mechanisms have been postulated. For example, HP activates a great variety of endogenous protective mediators including HIF-1α and HIF-2α stabilization increasing the capability of cell survival under severe oxygen deprivation (Wu et al., 2012, The protective role of hypoxic preconditioning in CNS, Anoxia, Dr. Pamela Padilla (Ed.), InTech, DOI: 10.5772/27621).
Brain diseases where hypoxia occurs mainly include stroke, cerebral palsy, traumatic injuries etc. Until now, there are no any effective drugs to protect brain from these diseases. Disclosure of the mechanism of HP will contribute to drug discovery for prevention against said diseases. A number of cellular adaptive responses to hypoxia are mediated by HIF-1α and activation of this factor by HP enhances the capability to tolerate severe anoxia or ischemia. The target genes of HIF-1, on the one hand, are involved in energy homeostasis, such as EPO in the regulation of erythropoiesis, vascular endothelial growth factor (VEGF) in angiogenesis, glucose transmitters (GLUTs) in glucose uptake and glycolytic enzymes of anaerobic glycolysis (Speer et al., Free Radio. Biol. Med. 2013 September; 62:23-36). Moreover, activation of HIF-1α in oligodendrocytes has been reported to induce EPO, which confers protection in oligodendrocytes against excitotoxicity (Sun et al., J. Neurosci. 2010 Jul. 14; 30(28):9621-30). In this sense the benefit of EPO in several diseases such as Multiple Sclerosis, stroke and traumatic brain injuries has been also demonstrated (Peng et al., J. Neurosurg. 2014 September; 121(3):653-64; Ehrenreich et al., Stroke 2009 December; 40(12): e647-56; Li et al., Ann. Neurol. 2004 December; 56(6):767-77). In addition, hypoximimetic agents (i.e. agents producing the same biological responses as the ones produced when hypoxia occurs) such as desferrioxamine (DFX) protect neuronal insults induced by 3-nitropoionic acid (Yang et al., J. Neurochem. 2005 May; 96(3):513-25). Therefore, PHDs inhibition by hypoximimetic small-molecules represents an interesting strategy or the development of neuroprotective therapies for the clinical management of conditions where hypoxia is present such as stroke, cerebral palsy, traumatic injuries and neurodegenerative diseases such as Multiple Sclerosis, Huntington disease, Alzheimer disease and Parkinson disease.
A substantial number of pharmacological studies (generally using nonspecific PDH2 inhibitors) have been conducted in animal models, and a few clinical studies have been performed. Indeed, several companies are involved in the discovery and development of PHD inhibitors for anemia and other indications such as IBD, myocardial ischaemia-reperfusion injury, acute lung injury, organ transplantation, acute kidney injury and arterial diseases are areas in which PHD inhibitors are actively being pursued by many researchers as a novel therapeutic approach (Rabinowitz M H, J. Med. Chem. 2013 Dec. 12; 56(23):9369-4025 and; Eltzschig et al., Nat. Rev. Drug Discov. 2014 November; 13(11):852-69).
The original description of HIF-selective PHDs as regulators of HIF expression has provided a template for the development of PHD-based molecular tools and therapies. Pharmacological inactivation of the PHDs by 2-OG analogues is sufficient to stabilize HIF-1α, but this action is nonspecific with respect to individual PHD isoforms and in vitro studies showed that the ODD sequence of HIF1αis hydroxylated most efficiently by PHD2 (Rabinowitz MH, J. Med. Chem. 2013 Dec. 12;56(23):9369-4025). These observations have generated considerable interest in identifying enzyme-modifying small-molecule inhibitors. Indeed, several PHD inhibitor classes have been described, including iron chelators such as DFX, hydralazine, AKB-4924, FG-2229, TM-6008 and 1-mimosine 1-mimosine; CUL2 deneddylators such as MLN4924; 2-OG mimics such as ximethyloxalylglycine and N-oxalyl-d-phenylalanine; PHD active-site blockers such as pyrazolopyridines, 8-hydroxyquinolines, compound A, FG-4497 and TM-6089; and Fe2+ substitutes such as Co2+, Ni2+ and Cu2+. The mechanism of action of these compounds is based on the observation that the binding of the co-substrate 2-OG to the catalytic domain, which harbours an essential Fe2+ ion, is crucial for enzymatic PHD2 activity. Therefore, chemical compounds that structurally mimic 2-OG, such as N-oxalylglycine or its precursor DMOG, inhibit PHD2 by blocking the entry of the co-substrate (Rabinowitz M H, J. Med. Chem. 2013 Dec. 12; 56(23):9369-4025 and; Eltzschig et al., Nat. Rev. Drug Discov. 2014 November; 13(11):852-69).
DFX is an hydroxamic acid derivative acting as a metal chelator that is in clinical use for the treatment of acute iron intoxication and of chronic iron overload due to transfusion-dependent anemias. However, DFX is not indicated for the treatment of chronic diseases and it is contraindicated in severe renal diseases since primarily the kidney excretes the drug and the iron chelate. Moreover, DFX only induces HIF-1α stabilization and activation at relatively high concentrations in the cells.
Triterpenoids are widely distributed in edible and medicinal plants and are an integral part of the human diet. Among them, pentacyclic triterpenoids are derived from the linear hydrocarbon squalene and they are highly multifunctional and, thus, have a wide range of commercial applications in the agriculture, food, cosmetics and pharmaceutical sectors as pesticides, drugs, adjuvants, antimicrobials, anticancer agents, surfactants, preservatives, etc. (Sheng et al., Nat. Prod. Rep. 2011 March; 28(3):543-93; Yadav et al., Toxins (Basel) 2010 October; 2(10):2428-66). The acidic function and hydroxyl (—OH) groups of the triterpenoids cannot interact with the stationary phase, as the two groups are located on the opposite sides of the compound. There are three main triterpene families: oleane, ursane, and lupane triterpenes. The main triterpenoids found in the oleane family are oleanolic acid, maslinic acid, erythrodiol, and β-amyrin; in the ursane family are ursolic acid and uvaol; and in the lupane family are lupeol, betulin, and betulinic acid (Yadav et al., Toxins (Basel) 2010 October; 2(10):2428-66).
Dietary triterpenoids such as oleanolic, ursolic, betulinic acid and maslinic acids have been extensively studied for bioactivity and pharmaceutical application and they have been widely used also as templates to produce novel derivatives with improved bioactivities. Triterpenoids have been shown to inhibit the activation of the transcription factors NF-κB (Nuclear factor-kappa B) and STAT3 (Signal transducer and activator of transcription 3), to modulate the Nrf2 (NFE2L2 or Nuclear factor (erythroid-derived 2)-like 2) pathway and to bind and activate the bile receptor TGR5 (Sato et al., Biochem. Biophys. Res. Comm. 2007 Nov. 3; 362(4):793-8). However, the potential activity of triterpenoids on PDH2 inhibition and HIF-1α stabilization has not been described. Surprisingly, ursolic, betulinic, and oleanolic acids, which are the precursors of compounds II to XIV disclosed herein bind to PHD2 but do not activate the HIF-1α pathway. This finding led to generate hydroxamate derivatives able to bind PHD2, to chelate Fe2+ in its active pocket and as a consequence these compounds activate the HIF pathway and stabilize HIF-1α and HIF-2α proteins. Compounds III, IV, V, VI, VIII, IX, X, XI, and XIV and XV are novel chemical entities whilst compounds II, VII, XII and XIII have been reported to have anticancer activity in vitro, which is not related to HIF-1α activation (Wiemann et al., Eur. J. Med. Chem. 2015 Dec. 1; 106:194-210; Wiemann et al., Bioorg. Med. Chem. Left. 2016 Feb. 1; 26(3):907-9; CN102180939 B). In fact, HIF-1α inhibition by some triterpenoids, as opposite to HIF-1α activation, has been suggested as a potential strategy for anti-cancer therapies (Dasgupta et al., Angiogenesis. 2015 July; 18(3): 283-99; Jin et al., Arch Pharm Res. 2007 April; 30(4): 412-8; Dai et al., J Nat Prod. 2006 December; 69(12): 1715-20).